The determination of oxybiotin in the presence of biotin.

The biological activity of oxybiotin (1, 2) and of an apparently similar substance (3) has been recently described. Prior to these reports the only compound possessing yeast growth activity comparable to that of biotin was desthiobiotin (4), which exerts its biological activity by virtue of the fact that it is converted into biotin by the yeast cell (5, 6). It was of importance to determine whether oxybiotin is likewise transformed into biotin by microorganisms or whether it has intrinsic activity. In order to investigate this point, an assay procedure was needed for the determination of oxybiotin in the presence of biotin. Since all of the organisms tested so far are stimulated by oxybiotin, it appeared unlikely that an organism could be found readily which would not respond to the oxygen analogue and thus provide the basis for a microbiological differential assay. A chemical method was therefore developed which is based on the different behavior of ethers and thio ethers toward oxidizing agents. It has been shown previously that potassium permanganate oxidizes biotin to the corresponding sulfone (7). This latter substance has a low degree of biotin activity in the presence of aspartic acid, and is inactive when this amino acid is not included in the medium (8). Oxybiotin could be expected to be stable toward dilute permanganate. It has now been observed that dilute potassium permanganate inactivates biotin completely in pure solution, as well as in extracts or hydrolysates of biological materials, while oxybiotin is virtually unaffected by such treatment. Microbiological assay of permanganate-treated materials makes it possible to determine their oxybiotin content. By the use of an assay procedure, based on the foregoing considerations, it has been demonstrated that Saccharomgces cerevisiae and Rhizobium trifolii utilize the oxybiotin molecule as such, and do not transform it into biotin.